The detection of single-stranded nucleic acid targets by hybridization to fluorescently labeled probes is of significant interest for the development of improved reagents for molecular diagnostics. Fluorescently labeled oligonucleotides also are useful probes of nucleic acid structure and hybridization at concentrations below those detectable by other non-isotopic analytical solution-phase methods. Morrison, L. E, and Stols, L. M., Biochemistry 32, 3095 (1993).
Cyanine dyes such as thiazole orange have demonstrated large fluorescence intensity increases upon binding to double stranded DNA. Makler, M. T., Lee, L. G., and Rectenwald, D. (1987) Cytometry 8, 568-570; Lee, L. G., Chen, C-H., and Chiu, L. A. (1986) Cytometry 7, 508-517; Lee, L. G. and Chen, C-H., U.S. Pat. No. 4,957,870 (Sep. 18, 1990) "Detection of Reticulocytes, RNA, and DNA"; Lee, L. G. and Chen, C-H., U.S. Pat. No. 4,883,867 (Nov. 28, 1989) "Detection of Reticulocytes, RNA, and DNA". This fluorescence intensity enhancement for thiazole orange has been estimated to be as high as 18,000. Glazer, A. N. and Rye, H. S., Nature 359, 859 (1992). Although covalently linked dye-oligonucleotide complexes have been used to configure assays based on fluorescence energy transfer and quenching, direct tethering of a cyanine dye to an oligonucleotide has not been accomplished to date.